PROJECT SUMMARY ABSTRACT Antibiotic-associated diarrhea (AAD) is a very common problem; approximately 50% of hospitalized patients receive antibiotics, and up to 25% of those patients develop AAD. While many AAD infectious etiologies, such as Clostridium difficile infection (CDI), have been well described, a recent study from Taiwan characterized Clostridium innocuum (CI) as a previously unrecognized cause of AAD with multidrug-resistance, including to vancomycin. This study suggested that CI causes gastrointestinal illness indistinguishable from CDI, which can include severe and pseudomembranous colitis, in both humans and an intestinal model of infection in mice. This recognition of the pathogenic potential of CI is in contrast to its prior classification as a clinically innocuous intestinal commensal. This was based on a single 1962 study that failed to demonstrate CI pathogenicity in a non-intestinal mouse model. Our preliminary clinical, microbiologic, and genomic data support potential reclassification of CI as an emerging pathogen. We have recently isolated CI from more than 20 children diagnosed with CDI (in whom the contribution of CI to illness is unclear) and one adult with recurrent AAD (in whom CI caused AAD). Further analysis suggests that many of these children were colonized with C. difficile and had another unidentified diarrheal etiology. In addition, several of our CI strains were noted to be positive for C. difficile toxins A/B by enzyme immunoassay (EIA). Thus, our preliminary data suggest a model whereby some CI strains have the ability to produce a protein that is recognized by C. difficile anti-toxin antibodies, and that CI may cause some cases of AAD, potentially related to this uncharacterized protein. To better characterize the epidemiologic significance and microbiologic characteristics of this emerging pathogen in US adults and children, we propose to: (1) Determine the clinical and molecular epidemiology of CI in symptomatic and asymptomatic adults and children; (2a) Identify the CI protein responsible for C. difficile toxin A/B EIA cross-reactivity, and (2b) Determine the association between this protein and CI pathogenicity. Because vancomycin use for CDI is increasing in the US, selective pressure may favor a rise in clinical infections with multidrug-resistant CI. Because of widespread use of C. difficile-specific diagnostic tests for CDI, diagnosis of clinically indistinguishable CI AAD will require substantial clinical microbiology practice change. For these reasons, it is imperative to understand the scope of CI AAD in the US. Successful completion of the proposed studies will guide future investigation of the pathogenesis, diagnosis, and clinical characteristics of CI infection. Our team and the Northwestern University academic environment possess the skills and resources necessary for successful completion of the proposed studies, including assembly of patient cohorts, microbiology, bacterial pathogenesis, proteomics, genomics, and bioinformatics.